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NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2018 Apr 13.
Published in final edited form as: Life Sci. 2014 Aug 2;113(1-2):45–54. doi: 10.1016/j.lfs.2014.07.031

Genome-wide analysis of DNA methylation in UVB- and DMBA/TPA-induced mouse skin cancer models

Anne Yuqing Yang a,b,c,1, Jong Hun Lee a,b,1, Limin Shu a,b, Chengyue Zhang a,b,c, Zheng-Yuan Su a,b, Yaoping Lu a,d, Mou-Tuan Huang a,d, Christina Ramirez b,d, Douglas Pung b, Ying Huang a,b,c, Michael Verzi f, Ronald P Hart g, Ah-Ng Tony Kong a,b,d,*
PMCID: PMC5897904  NIHMSID: NIHMS954849  PMID: 25093921

Abstract

Aims

Ultraviolet irradiation and carcinogens have been reported to induce epigenetic alterations, which potentially contribute to the development of skin cancer. We aimed to study the genome-wide DNA methylation profiles of skin cancers induced by ultraviolet B (UVB) irradiation and 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-1,3-acetate (TPA).

Main methods

Methylated DNA immunoprecipitation (MeDIP) followed by next-generation sequencing was utilized to ascertain the DNA methylation profiles in the following common mouse skin cancer models: SKH-1 mice treated with UVB irradiation and CD-1 mice treated with DMBA/TPA. Ingenuity® pathway analysis (IPA) software was utilized to analyze the data and to identify gene interactions among the different pathways.

Key findings

6,003 genes in the UVB group and 5,424 genes in the DMBA/TPA group exhibited a greater than 2-fold change in CpG methylation as mapped by the IPA software. The top canonical pathways identified by IPA after the two treatments were ranked were pathways related to cancer development, cAMP-mediated signaling, G protein-coupled receptor signaling and PTEN signaling associated with UVB treatment, whereas protein kinase A signaling and xenobiotic metabolism signaling were associated with DMBA/TPA treatment. In addition, the mapped IL-6-related inflammatory pathways displayed alterations in the methylation profiles of inflammation-related genes linked to UVB treatment.

Significance

Genes with altered methylation were ranked in the UVB and DMBA/TPA models, and the molecular interaction networks of those genes were identified by the IPA software. The genome-wide DNA methylation profiles of skin cancers induced by UV irradiation or by DMBA/TPA will be useful for future studies on epigenetic gene regulation in skin carcinogenesis.

Keywords: DNA methylation, epigenetics, MeDIP-Seq, UVB, DMBA/TPA

Introduction

Accumulating evidence suggests that epigenetic DNA alterations play a crucial role in cancer initiation and development. Specifically, aberrant DNA methylation at the 5′-position of cytosine in CG dinucleotides in the cancer genome is postulated to be the most relevant epigenetic change in cancer [1]. DNA methylation affects gene expression and therefore regulates a wide range of biological processes, including proliferation, cell death, mutation and cancer initiation/promotion/progression [2, 3]. Both global hypomethylation and regional hypermethylation are characteristics of tumorigenesis [4, 5]. DNA hypermethylation at promoter regions is a predominant epigenetic mechanism for reducing the expression of tumor suppressor genes. Epigenetic silencing of tumor suppressor genes has been reported in mouse models of malignant tumors [6, 7]. Thus, the present study aimed to profile the global DNA methylation changes that occur during carcinogenesis. To address this aim, we conducted global profiling of DNA methylation changes in two representative skin carcinogenesis models, ultraviolet B (UVB)-exposed SKH-1 hairless mice and DMBA/TPA-induced carcinogenesis in CD-1 mice.

Ultraviolet irradiation has been reported to induce epigenetic alterations, which may contribute to the development of skin cancer [8]. However, the precise mechanism by which UV irradiation is related to melanomagenesis remains unclear. In addition, a method for screening epigenetically modified genes in melanoma patients needs to be established [9, 10]. Mutations in oncogenes and tumor suppressor genes have been reported in melanoma. However, most of these mutations are not present in non-melanoma skin cancer [11], suggesting a specific role for UV irradiation in melanomagenesis. In skin tumors, UV irradiation has been reported to induce epigenetic modifications and may contribute to the development of skin cancer. Epigenetic changes, such as DNA methylation and histone modification, may play a crucial role in the initiation and development of certain types of cancer [12]. Epigenetic alterations generally represent the interface between the environment and the genome [13].

The multistage skin carcinogenesis model is established by applying a sub-threshold dose of the carcinogen DMBA followed by repetitive treatments with the tumor promoter TPA. With three well-defined stages—initiation, promotion, and progression—this model is similar to natural human tumor development. This model has been widely used to investigate the anti-tumor efficacy of chemicals and the molecular events that occur during each stage of tumor development. Several genetic alterations identified in human skin cancer patients have also been described in the mouse multistage skin carcinogenesis model, such as changes in cyclin D1 [14], TP53 [15], CDKN21 [16] and PTEN [17]. The underlying similarity in the biology of cancer between mice and humans implies that genes related to mouse tumor development may also be relevant to human tumor development.

In the present study, we used methylated DNA immunoprecipitation (MeDIP) coupled with next-generation sequencing to profile the whole genome DNA methylation patterns from the UVB and DMBA/TPA models. The MeDIP-Seq results were evaluated by Ingenuity® Pathway Analysis (IPA) to investigate genetic crosstalk and signal/function overlap. The present study included an initial assessment of genes with a modified methylation profile and the identification of interacting molecular networks in skin carcinogenesis models.

Materials and methods

Chemicals and antibodies

The chemicals used in the current study were as follows: 7,12-dimethylbenz(a)anthracene (DMBA; Sigma-Aldrich, MO, USA) and 12-O-tetradecanoylphorbol-1,3-acetate (TPA; Alexis Co., CA, USA). The 5-methylcytidine monoclonal antibody was purchased from Eurogentec., Belgium.

Mice and skin cancer induction

Two representative skin carcinogenesis models were utilized in the present study. SKH-1 hairless female mice, 7-8 weeks old, were treated with UVB (30 mJ/cm2) twice a week for 36 weeks. The UV lamps (FS72T12-UVB-HO; National Biological, Twinsburg, OH) emitted UVB (280-320 nm; 70-80% of the total energy) and ultraviolet A (320-375 nm; 20-75% of the total energy). The dose of UVB was quantified using a UVB Spectra 305 dosimeter (The Daavlin Company, Bryan, OH). The radiation was further calibrated using a research radiometer/photometer (model IL-1700; International Light Inc., Newburyport, MA). The mice were assessed twice weekly for the appearance of papillomas and carcinomas. Skin papilloma and carcinoma samples were collected, frozen in liquid nitrogen and stored at −80 °C. The epidermises of age-matched untreated mice were isolated from fresh skin as a control group.

Six-week-old female CD-1 mice were used for the DMBA/TPA-induced multistage carcinogenesis model. One day before treatment, the backs of the mice were shaved. For tumor initiation, 200 nmol DMBA in 200 μL of acetone was injected into the back skin of the mice. Three days after DMBA treatment, 5 nmol TPA in 200 μL of acetone was applied three times a week for 11 weeks to induce tumor promotion and progression.

Global analysis of methylated DNA

Genomic DNA (gDNA) was extracted from UV irradiation-induced tumor samples from 3 female mice and from non-irradiated epidermis samples from 3 female age-matched SKH-1 mice; from female CD-1 mice in the DMBA/TPA-induced carcinogenesis model for the MeDIP-Seq assay. gDNA was extracted using a DNeasy Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. The gDNA was electrophoresed on an agarose gel, and the OD ratios were determined to confirm the purity and concentration of the gDNA prior to fragmentation by Covaris (Covaris, Inc., Woburn, MA USA). Fragmented gDNA was evaluated for size distribution and concentration using an Agilent Bioanalyzer 2100 and a NanoDrop spectrophotometer.

MeDIP-Seq

MeDIP was performed to analyze genome-wide methylation. MeDIP was performed using a MagMeDIP Kit from Diagenode according to the manufacturer’s instructions. Methylated DNA was recovered by immunoprecipitation with antibodies specific to methylated cytosine used to separate methylated DNA fragments from unmethylated fragments, and Illumina libraries were created from the captured gDNA using NEBNext reagents (catalog# E6040; New England Biolabs, Ipswich, MA, USA). Enriched libraries were evaluated for size distribution and concentration using an Agilent Bioanalyzer 2100. The samples were then sequenced on an Illumina HiSeq2000 machine, which generated paired-end reads of 90 or 100 nucleotides (nt). The results were analyzed for data quality and exome coverage using the platform provided by DNAnexus (DNAnexus, Inc., Mountain View, CA, USA). Samples were sent to Otogenetics Corporation (Norcross, GA) for Illumina sequencing and alignment with the genome. The resulting BAM files were downloaded for analysis.

MeDIP alignments were compared with control samples using Cuffdiff 2.0.2 with no length correction [18]. Briefly, a list of overlapping regions of sequence alignment common to both the immunoprecipitated and the control samples was created and used to judge the quantitative enrichment in MeDIP samples over control samples using Cuffdiff; statistically significant peaks at 5% FDR and a minimum 4-fold difference using the Cummerbund package in R were selected [18]. Peaks were matched with adjacent annotated genes using ChIPpeakAnno [19].

Functional and pathway analysis by Ingenuity Pathway Analysis (IPA)

We analyzed lists of genes with significant fold changes (based on the P values; UV-induced tumor vs. control and DMBA/TPA treatment vs. control) in the methylation pattern (increases and decreases) ascertained in the MeDIP-Seq experiment using Ingenuity® Pathways Analysis 4.0 (IPA 4.0, Ingenuity Systems, www.ingenuity.com). IPA utilized gene symbols that were identified as neighboring enriched methylation peaks by ChIPpeakAnno used for all of the analyses. IPA mapped 6,003 genes in the UVB group and 5,424 genes in the DMBA/TPA group with a ≥ 2-fold change compared with the control correspondingly. Based on these fold change data, IPA identified biological functions and canonical pathways related to UVB-induced cancer. The list of genes within canonical pathways was ranked using the ratio of the number of genes mapped to each pathway to the total number of genes in the corresponding pathway, which is presented as observations/total in Table 3.

Table 3.

Top 5 altered canonical pathways determined using Ingenuity Pathways Software in the UVB and DMBA/TPA groups. The shared pathways are shown in bold.

Rank Name Ratio Observation/Total p-value
UVB/Control
 1 cAMP-mediated signaling 0.478 108/226 1.27E-09
 2 G-Protein Coupled Receptor Signaling 0.442 122/276 5.24E-09
3 Molecular Mechanisms of Cancer 0.371 144/388 9.86E-07
 4 PTEN Signaling 0.42 58/138 5.21E-06
 5 Role of Osteoblasts, Osteoclasts and Chondrocytes in Rheumatoid Arthritis 0.388 97/250 5.58E-06
DMBA-TPA/Control
 1 Protein Kinase A Signaling 0.352 144/409 5.5E-06
2 Molecular Mechanisms of Cancer 0.325 126/338 4.57E-05
 3 Xenobiotic Metabolism Signaling 0.351 101/288 7.59E-05
 4 Regulation of the Epithelial-Mesenchymal Transition Pathway 0.342 67/196 0.0011
 5 Mouse Embryonic Stem Cell Pluripotency 0.394 39/99 0.0013

Results

MeDIP-Seq results

Skin samples were collected from the UVB- and DMBA/TPA-induced mouse skin cancer models, and gDNA was isolated from each sample. A whole-genome DNA methylation analysis was performed on the DNA samples using the described MeDIP-Seq method. The results were analyzed in a paired manner comparing the tumor to the normal skin tissue samples for each model.

In the UVB group, 6,003 genes were identified in the two samples with a ≥ 2-fold change in methylation and an increase or decrease in gene expression. Compared with the control, 4,140 genes exhibited increased methylation, and methylation was decreased in 1,863 genes (Figure 1). However, in the DMBA/TPA treatment group, 5,424 genes were identified with a ≥ 2-fold change in peak reads between the tumor and the normal skin samples. Among these 5,424 genes, the methylation pattern was up-regulated in 3,781 genes and down-regulated in 1,643 genes. To prioritize those changes, we ranked the top 50 up-regulated (+, Table 1) and down-regulated (−, Table 2) genes based on the log2 fold change from highest to lowest, all with P values less than 0.05.

Figure 1.

Figure 1

Total number of significantly up-regulated and down-regulated genes based on changes in methylation (≥ 2-fold change) in the UVB and DMBA/TPA groups.

Table 1.

Top 50 annotated genes with up-regulated methylation ranked by log2 fold change. (A) UVB group. (B) DMBA/TPA group.

(A)
Rank Symbol Gene Name Log2 Fold Change (UVB/Control) Location Type(s)
1 RBFOX1 RNA binding protein, fox-1 homolog (C. elegans) 1 5.457 Cytoplasm other
2 IMPG2 interphotoreceptor matrix proteoglycan 2 5.367 Extracellular Space other
3 DGKK diacylglycerol kinase, kappa 5.31 Cytoplasm kinase
4 MAD1L1 MAD1 mitotic arrest deficient-like 1 (yeast) 5.252 Nucleus other
5 EVX2 even-skipped homeobox 2 5.19 Nucleus transcription regulator
6 PAN3 PAN3 poly(A) specific ribonuclease subunit homolog (S. cerevisiae) 4.989 Cytoplasm other
7 AAMP angio-associated, migratory cell protein 4.837 Plasma Membrane other
8 ARHGAP18 Rho GTPase activating protein 18 4.837 Cytoplasm other
9 ACAA2 acetyl-CoA acyltransferase 2 4.574 Cytoplasm enzyme
10 OLFM1 olfactomedin 1 4.574 Cytoplasm other
11 TGS1 trimethylguanosine synthase 1 4.574 Nucleus enzyme
12 DYM dymeclin 4.525 Cytoplasm other
13 Hspg2 heparan sulfate proteoglycan 2 4.474 Extracellular Space other
14 Kcnip2 Kv channel-interacting protein 2 4.474 Plasma Membrane other
15 TNS1 tensin 1 4.474 Plasma Membrane other
16 AGAP1 ArfGAP with GTPase domain, ankyrin repeat and PH domain 1 4.367 Cytoplasm enzyme
17 CCDC180 coiled-coil domain containing 180 4.367 Other other
18 EDN1 endothelin 1 4.367 Extracellular Space cytokine
19 FOXE1 forkhead box E1 (thyroid transcription factor 2) 4.367 Nucleus transcription regulator
20 KCNN4 potassium intermediate/small conductance calcium-activated channel, subfamily N, member 4 4.367 Plasma Membrane ion channel
21 LOXHD1 lipoxygenase homology domains 1 4.367 Extracellular Space other
22 DCP2 decapping mRNA 2 4.252 Nucleus enzyme
23 DET1 de-etiolated homolog 1 (Arabidopsis) 4.252 Nucleus other
24 DSC3 desmocollin 3 4.252 Plasma Membrane other
25 HOXD11 homeobox D11 4.252 Nucleus transcription regulator
26 MAML2 mastermind-like 2 (Drosophila) 4.252 Nucleus transcription regulator
27 MYO1E myosin IE 4.252 Cytoplasm enzyme
28 PCBD1 pterin-4 alpha-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor 1 alpha 4.252 Nucleus transcription regulator
29 WNT3 wingless-type MMTV integration site family, member 3 4.252 Extracellular Space other
30 CENPF centromere protein F, 350/400kDa 4.126 Nucleus other
31 Dos downstream of Stk11 4.126 Other other
32 FBXO11 F-box protein 11 4.126 Cytoplasm enzyme
33 GPR37 G protein-coupled receptor 37 (endothelin receptor type B-like) 4.126 Plasma Membrane G-protein coupled receptor
34 HPCA hippocalcin 4.126 Cytoplasm other
35 LIMS2 LIM and senescent cell antigen-like domains 2 4.126 Cytoplasm other
36 LRRC8B leucine rich repeat containing 8 family, member B 4.126 Other other
37 LTA4H leukotriene A4 hydrolase 4.126 Cytoplasm enzyme
38 MEMO1 mediator of cell motility 1 4.126 Cytoplasm other
39 mir-221 microRNA 221 4.126 Cytoplasm microRNA
40 mir-802 microRNA 802 4.126 Cytoplasm microRNA
41 Olfr1323 olfactory receptor 1323 4.126 Plasma Membrane G-protein coupled receptor
42 PLN phospholamban 4.126 Cytoplasm transporter
43 PTPN23 protein tyrosine phosphatase, non-receptor type 23 4.126 Cytoplasm phosphatase
44 SLC7A9 solute carrier family 7 (amino acid transporter light chain, bo,+ system), member 9 4.126 Plasma Membrane transporter
45 VRK1 vaccinia related kinase 1 4.126 Nucleus kinase
46 ZBTB34 zinc finger and BTB domain containing 34 4.126 Nucleus other
47 ZNF622 zinc finger protein 622 4.126 Nucleus other
48 SMYD2 SET and MYND domain containing 2 4.082 Cytoplasm enzyme
49 DYSF dysferlin, limb girdle muscular dystrophy 2B (autosomal recessive) 3.989 Plasma Membrane other
50 Ear2 (includes others) eosinophil-associated, ribonuclease A family, member 2 3.989 Cytoplasm enzyme
(B)
Rank Symbol Gene Name Log2 Fold Change (DMBA-TPA/Control) Location Type(s)
1 FAM135A family with sequence similarity 135, member A 5.974 Other enzyme
2 CADM2 cell adhesion molecule 2 5.231 Plasma Membrane other
3 VWC2L von Willebrand factor C domain containing protein 2-like 4.877 Extracellular Space other
4 PTH2R parathyroid hormone 2 receptor 4.708 Plasma Membrane G-protein coupled receptor
5 NPY neuropeptide Y 4.515 Extracellular Space other
6 TNS1 tensin 1 4.408 Plasma Membrane other
7 PHYHIPL phytanoyl-CoA 2-hydroxylase interacting protein-like 4.408 Cytoplasm other
8 COX7C cytochrome c oxidase subunit VIIc 4.408 Cytoplasm enzyme
9 CMYA5 cardiomyopathy associated 5 4.408 Plasma Membrane other
10 HELB helicase (DNA) B 4.351 Nucleus enzyme
11 WDR63 WD repeat domain 63 4.292 Other other
12 SIPA1L2 signal-induced proliferation-associated 1 like 2 4.292 Other other
13 let-7 microRNA let-7a-1 4.292 Cytoplasm microRNA
14 DSEL dermatan sulfate epimerase-like 4.292 Extracellular Space enzyme
15 ZNF521 zinc finger protein 521 4.167 Nucleus other
16 TMTC2 transmembrane and tetratricopeptide repeat containing 2 4.167 Cytoplasm other
17 OTOL1 otolin 1 4.167 Other other
18 METTL21A methyltransferase like 21A 4.167 Other enzyme
19 INTU inturned planar cell polarity protein 4.167 Cytoplasm other
20 DHCR7 7-dehydrocholesterol reductase 4.167 Cytoplasm enzyme
21 Cyp2a12/Cyp2a22 cytochrome P450, family 2, subfamily a, polypeptide 12 4.167 Cytoplasm enzyme
22 CTSC cathepsin C 4.167 Cytoplasm peptidase
23 CHORDC1 cysteine and histidine-rich domain (CHORD) containing 1 4.167 Other other
24 CBLN1 cerebellin 1 precursor 4.167 Cytoplasm other
25 THAP1 THAP domain containing, apoptosis associated protein 1 4.029 Nucleus other
26 RYBP RING1 and YY1 binding protein 4.029 Nucleus transcription regulator
27 NDUFA12 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 12 4.029 Cytoplasm enzyme
28 Mug1/Mug2 murinoglobulin 1 4.029 Extracellular Space transporter
29 MFSD10 major facilitator superfamily domain containing 10 4.029 Other transporter
30 KIF16B kinesin family member 16B 4.029 Cytoplasm enzyme
31 GRID2 glutamate receptor, ionotropic, delta 2 4.029 Plasma Membrane ion channel
32 Gm4836 (includes others) predicted gene 4836 4.029 Cytoplasm other
33 FOXN3 forkhead box N3 4.029 Nucleus transcription regulator
34 Cyp4f16/Gm9705 cytochrome P450, family 4, subfamily f, polypeptide 16 4.029 Cytoplasm enzyme
35 CXorf22 chromosome X open reading frame 22 4.029 Other other
36 Pag1 phosphoprotein associated with glycosphingolipid microdomains 1 3.955 Plasma Membrane other
37 MSH6 mutS homolog 6 3.923 Nucleus enzyme
38 ZFAT zinc finger and AT hook domain containing 3.877 Nucleus other
39 Vmn1r188 (includes others) vomeronasal 1 receptor 217 3.877 Plasma Membrane G-protein coupled receptor
40 TYW3 tRNA-yW synthesizing protein 3 homolog (S. cerevisiae) 3.877 Other other
41 TPO thyroid peroxidase 3.877 Plasma Membrane enzyme
42 TLR4 toll-like receptor 4 3.877 Plasma Membrane transmembrane receptor
43 THSD7B thrombospondin, type I, domain containing 7B 3.877 Other other
44 SPINK5 serine peptidase inhibitor, Kazal type 5 3.877 Extracellular Space other
45 SLC9A8 solute carrier family 9, subfamily A (NHE8, cation proton antiporter 8), member 8 3.877 Cytoplasm transporter
46 SEC23A Sec23 homolog A (S. cerevisiae) 3.877 Cytoplasm transporter
47 RPS4Y1 ribosomal protein S4, Y-linked 1 3.877 Cytoplasm other
48 PPP1R1C protein phosphatase 1, regulatory (inhibitor) subunit 1C 3.877 Cytoplasm phosphatase
49 PARP8 poly (ADP-ribose) polymerase family, member 8 3.877 Other other
50 NLRP4 NLR family, pyrin domain containing 4 3.877 Cytoplasm other

Table 2.

Top 50 annotated genes with down-regulated methylation ranked by log2 fold change. (A) UVB group. (B) DMBA/TPA group.

(A)
Symbol Gene Name Log2 Fold Change (UVB/Control) Location Type(s)
1 Nrxn3 neurexin III −4.543 Plasma Membrane other
2 SYN2 synapsin II −4.378 Plasma Membrane other
3 Mup1 (includes others) major urinary protein 1 −4.333 Extracellular Space other
4 KRT86 keratin 86 −4.24 Cytoplasm other
5 SULT1C3 sulfotransferase family, cytosolic, 1C, member 3 −4.24 Cytoplasm enzyme
6 ATP1A3 ATPase, Na+/K+ transporting, alpha 3 polypeptide −4.191 Plasma Membrane transporter
7 CHORDC1 cysteine and histidine-rich domain (CHORD) containing 1 −4.191 Other other
8 NRXN1 neurexin 1 −4.191 Plasma Membrane transporter
9 Htr6 5-hydroxytryptamine (serotonin) receptor 6, G protein-coupled −4.141 Plasma Membrane G-protein coupled receptor
10 PAM peptidylglycine alpha-amidating monooxygenase −4.141 Plasma Membrane enzyme
11 SERPINB3 serpin peptidase inhibitor, clade B (ovalbumin), member −4.141 Cytoplasm other
12 TDRD3 tudor domain containing 3 −4.141 Nucleus transcription regulator
13 Olfr1153 olfactory receptor 1153 −4.088 Plasma Membrane G-protein coupled receptor
14 WFIKKN2 WAP, follistatin/kazal, immunoglobulin, kunitz and netrin domain containing 2 −4.088 Other other
15 ABAT 4-aminobutyrate aminotransferase −4.034 Cytoplasm enzyme
16 ARHGAP6 Rho GTPase activating protein 6 −4.034 Cytoplasm other
17 AUH AU RNA binding protein/enoyl-CoA hydratase −4.034 Cytoplasm enzyme
18 CEP70 centrosomal protein 70kDa −4.034 Cytoplasm other
19 DGKH diacylglycerol kinase, eta −4.034 Cytoplasm kinase
20 GRIA1 glutamate receptor, ionotropic, AMPA 1 −4.034 Plasma Membrane ion channel
21 SLC4A7 solute carrier family 4, sodium bicarbonate cotransporter, member 7 −4.034 Plasma Membrane transporter
22 SLITRK1 SLIT and NTRK-like family, member 1 −4.034 Other other
23 Speer4a (includes others) spermatogenesis associated glutamate (E)-rich protein 4a −4.034 Nucleus other
24 CETN1 centrin, EF-hand protein, 1 −3.977 Nucleus enzyme
25 CYLC1 cylicin, basic protein of sperm head cytoskeleton 1 −3.977 Cytoplasm other
26 HS3ST5 heparan sulfate (glucosamine) 3-O-sulfotransferase 5 −3.977 Cytoplasm enzyme
27 Ott (includes others) ovary testis transcribed −3.977 Other other
28 Scg5 secretogranin V −3.977 Cytoplasm other
29 CHM choroideremia (Rab escort protein 1) −3.918 Cytoplasm enzyme
30 Clvs2 clavesin 2 −3.918 Cytoplasm other
31 MRPS30 mitochondrial ribosomal protein S30 −3.918 Cytoplasm enzyme
32 Olfr1010 olfactory receptor 1010 −3.918 Plasma Membrane G-protein coupled receptor
33 PPP1R12A protein phosphatase 1, regulatory subunit 12A −3.918 Cytoplasm phosphatase
34 ZMYND11 zinc finger, MYND-type containing 11 −3.918 Nucleus other
35 ADCY10 adenylate cyclase 10 (soluble) −3.857 Cytoplasm enzyme
36 Agtr1b angiotensin II receptor, type 1b −3.857 Plasma Membrane G-protein coupled receptor
37 APAF1 apoptotic peptidase activating factor 1 −3.857 Cytoplasm other
38 AQP1 aquaporin 1 −3.857 Plasma Membrane transporter
39 CDH10 cadherin 10, type 2 (T2-cadherin) −3.857 Plasma Membrane other
40 CHRM3 cholinergic receptor, muscarinic 3 −3.857 Plasma Membrane G-protein coupled receptor
41 GCNT2 glucosaminyl (N-acetyl) transferase 2, I-branching enzyme (I blood group) −3.857 Cytoplasm enzyme
42 GNAI1 guanine nucleotide binding protein (G protein), alpha inhibiting activity polypeptide 1 −3.857 Plasma Membrane enzyme
43 SELENBP1 selenium binding protein 1 −3.857 Cytoplasm other
44 SP110 SP110 nuclear body protein −3.857 Nucleus other
45 TMEM5 transmembrane protein 5 −3.857 Plasma Membrane other
46 XIRP2 xin actin-binding repeat containing 2 −3.857 Other other
47 ZKSCAN2 zinc finger with KRAB and SCAN domains 2 −3.857 Nucleus transcription regulator
48 1700009N14Rik RIKEN cDNA 1700009N14 gene −3.793 Other transporter
49 AGPAT9 1-acylglycerol-3-phosphate O-acyltransferase 9 −3.793 Cytoplasm enzyme
50 ASCL1 achaete-scute complex homolog 1 (Drosophila) −3.793 Nucleus transcription regulator
(B)
Rank Symbol Gene Name Log2 Fold Change (DMBA-TPA/Control) Location Type(s)
1 EBPL emopamil binding protein-like −5.292 Cytoplasm enzyme
2 PANX1 pannexin 1 −5.247 Plasma Membrane transporter
3 HES5 hairy and enhancer of split 5 (Drosophila) −4.632 Nucleus other
4 LHX4 LIM homeobox 4 −4.247 Nucleus transcription regulator
5 GSG1L GSG1-like −4.247 Plasma Membrane other
6 PBX1 pre-B-cell leukemia homeobox 1 −4.199 Nucleus transcription regulator
7 LRRC8B leucine rich repeat containing 8 family, member B −4.199 Other other
8 ASAH2 N-acylsphingosine amidohydrolase (non-lysosomal ceramidase) 2 −4.199 Cytoplasm enzyme
9 ALKBH3 alkB, alkylation repair homolog 3 (E. coli) −4.199 Nucleus enzyme
10 ZNF518B zinc finger protein 518B −4.100 Other other
11 MAN1A1 mannosidase, alpha, class 1A, member 1 −4.100 Cytoplasm enzyme
12 TOX3 TOX high mobility group box family member 3 −3.993 Other other
13 LSM11 LSM11, U7 small nuclear RNA associated −3.936 Nucleus other
14 TCEA3 transcription elongation factor A (SII), 3 −3.877 Nucleus transcription regulator
15 OR7D2 olfactory receptor, family 7, subfamily D, member 2 −3.877 Plasma Membrane G-protein coupled receptor
16 KIAA0947 KIAA0947 −3.877 Other other
17 CBLB Cbl proto-oncogene B, E3 ubiquitin protein ligase −3.877 Nucleus other
18 NOS1AP nitric oxide synthase 1 (neuronal) adaptor protein −3.816 Cytoplasm other
19 MACC1 metastasis associated in colon cancer 1 −3.816 Nucleus other
20 ZNF277 zinc finger protein 277 −3.752 Nucleus transcription regulator
21 Sp100 nuclear antigen Sp100 −3.752 Nucleus transcription regulator
22 KCNA6 potassium voltage-gated channel, shaker-related subfamily, member 6 −3.752 Plasma Membrane ion channel
23 C19orf10 chromosome 19 open reading frame 10 −3.752 Extracellular Space cytokine
24 TMEM17 transmembrane protein 17 −3.685 Extracellular Space other
25 FAM92B family with sequence similarity 92, member B −3.650 Other other
26 TBC1D5 TBC1 domain family, member 5 −3.646 Extracellular Space other
27 Nrg1 neuregulin 1 −3.614 Extracellular Space growth factor
28 GORASP1 golgi reassembly stacking protein 1, 65kDa −3.614 Cytoplasm other
29 AHRR aryl-hydrocarbon receptor repressor −3.614 Nucleus other
30 ADORA2A adenosine A2a receptor −3.614 Plasma Membrane G-protein coupled receptor
31 RAB11A RAB11A, member RAS oncogene family −3.540 Cytoplasm enzyme
32 ISY1-RAB43 ISY1-RAB43 readthrough −3.540 Nucleus other
33 GNE glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase −3.540 Cytoplasm kinase
34 FAM98A family with sequence similarity 98, member A −3.540 Other other
35 ENPP4 ectonucleotide pyrophosphatase/phosphodiesterase 4 (putative) −3.540 Other enzyme
36 CCDC43 coiled-coil domain containing 43 −3.540 Other other
37 ARHGEF10 Rho guanine nucleotide exchange factor (GEF) 10 −3.540 Cytoplasm enzyme
38 TARSL2 threonyl-tRNA synthetase-like 2 −3.462 Other enzyme
39 SCARB1 scavenger receptor class B, member 1 −3.462 Plasma Membrane transporter
40 RAB27A RAB27A, member RAS oncogene family −3.462 Cytoplasm enzyme
41 L3MBTL3 l(3)mbt-like 3 (Drosophila) −3.462 Nucleus other
42 Higd1a HIG1 domain family, member 1A −3.462 Cytoplasm other
43 GRIA1 glutamate receptor, ionotropic, AMPA 1 −3.462 Plasma Membrane ion channel
44 GPC5 glypican 5 −3.462 Plasma Membrane other
45 CLGN calmegin −3.462 Cytoplasm peptidase
46 CHKA choline kinase alpha −3.462 Cytoplasm kinase
47 ASGR1 asialoglycoprotein receptor 1 −3.462 Plasma Membrane transmembrane receptor
48 AMY2A amylase, alpha 2A (pancreatic) −3.462 Extracellular Space enzyme
49 C1orf109 chromosome 1 open reading frame 109 −3.444 Other other
50 CADPS Ca++-dependent secretion activator −3.430 Plasma Membrane other

Pathway analysis by IPA

To ascertain the significance of the methylation changes, 6,003 genes in the UVB group and 5,424 genes in the DMBA/TPA group with a greater than 2-fold change in methylation were analyzed using the Ingenuity® Pathway Analysis (IPA) software package. The top 5 canonical signaling pathways were categorized (Table 3) based on the ratio of the number of input genes to the total number of genes in the corresponding pathway in the Ingenuity® Pathway Analysis software. Fisher’s exact test was used to calculate P values to determine the significance of the associations of the input genes with the canonical pathways.

IPA identified more than 50 signaling pathways containing genes with significantly up-regulated and down-regulated methylation. The interleukin-6 (IL-6)-related signaling pathway was mapped by IPA to the UVB group (Figure 2); the methylation profile is presented in red (up-regulation) and green (down-regulation). Figure 2B lists the genes involved in the IL-6 signaling pathway that exhibited altered methylation (14 up-regulated and 30 down-regulated) as mapped by IPA. It has been reported that IL-6 protein expression is induced by UV irradiation [20]. Correspondingly, the MeDIP-Seq data revealed that IL-6 up-regulated methylation by 3.252-fold (log2). Based on the IPA functional pathways, skin cancer belongs to the category of mechanism of cancer. The top 5 genes related to skin cancer based on IPA are ranked by fold change in Table 4. This list includes genes with increased methylation and genes with decreased methylation that are related to skin cancer pathways by IPA.

Figure 2.

Figure 2

(A) Genes mapped to the IL-6 pathway by IPA Software. Red, increased methylation; green, decreased methylation; UVB irradiated vs. control. (B) List of genes mapped to the IL-6 pathway by IPA.

Table 4.

Top 5 genes with altered methylation (up-regulated or down-regulated) that were related to skin cancer using the IPA Software Functional and Diseases analysis module in the UVB and the DMBA/TPA groups. The shared genes are shown in bold.

Rank Mapped Genes Log2 Fold Change
UVB/Control
Up-regulated
 1 RBFOX1 5.457
 2 LOXHD1 4.367
 3 EDN1 4.367
 4 DYSF 3.989
 5 NPSR1 3.837
Down-regulated
 1 SULT1C3 −4.24
 2 ABAT −4.034
3 GRIA1 −4.034
 4 PCSK1 −3.726
 5 SCN2A −3.655
DMBA-TPA/Control
Up-regulated
 1 GRID2 4.029
 2 NLRP4 3.877
 3 EMR1 3.877
 4 IL15 3.877
 5 PCDH18 3.708
Down-regulated
1 GRIA1 −3.462
 2 CADPS −3.43
 3 BCL2L11 −3.292
 4 ACVR1C −3.292
 5 GRM3 −3.292

Discussion

It was previously reported that hypermethylation of CpG islands in tumor suppressor genes occurs in human squamous cell carcinoma cell lines and in primary skin tumor tissues. Similar changes in DNA methylation patterns were observed in the multistage mouse skin cancer model. In addition, loss of global genomic methylation has been shown to be associated with increased aggressiveness of mouse skin cancer cell lines. However, the precise mechanism by which UV irradiation promotes melanoma remains unclear. Furthermore, there is no method for screening potential epigenetically modified genes involved in promoting skin cancers [10, 2123]. Long-term exposure to UV irradiation is considered a major etiologic risk factor for the development of melanoma and non-melanoma skin cancers. Epigenetic alterations are generally considered to represent the interface between the environment and the genome [13]. Ultraviolet irradiation has been reported to induce epigenetic alterations, which may contribute to the development of skin cancer. In the present study, we performed global genome methylation screening using MeDIP-Seq to identify genomic loci with aberrant methylation patterns in cancer tissues.

One of the adverse effects of UV irradiation that has been observed in skin tumor development is a chronic and sustained inflammatory response. The relationship between inflammation and epigenetic modifications in cancer is under active investigation [2426]. In this study, a UV irradiation-induced abnormal inflammatory response was suggested based on the IPA mapped IL-6 pathways, which included the methylation profiles of pro-inflammatory cytokines, receptors and mitogen-activated protein kinases. Higher levels of pro-inflammatory cytokines are associated with tumor development and progression [27, 28]. Interleukins (ILs) have different systemic functions and are involved in inflammation. Nile et al. reported that the methylation of CpGs in the promoter region of IL-6 affected the mRNA levels in mononuclear cells [24]. Tekpli et al. reported that the methylation of CpGs near the IL-6 transcriptional start site is significantly higher in non-small cell lung cancer cells and is associated with lower IL-6 mRNA expression [25]. Our study demonstrated that IL-6 gene methylation was significantly higher in UV irradiation-induced skin tumors.

IL-6-Jak-Stat3 inflammatory signaling is also involved in cell survival and provides a proliferative advantage in the two-stage chemical carcinogenesis model using DMBA as the tumor initiator and TPA as the promoter. Phosphorylated-Stat3 overexpression in a papilloma cell line leads to enhanced cell migration and invasion [29]. Transgenic mice with constitutive Stat3 expression have a shorter latency period and increased tumor incidence compared with non-transgenic littermates after DMBA/TPA treatment [30]. Moreover, mice with constitutively activated Stat3 bypassed the premalignant stage and were initially diagnosed with carcinoma in situ, which rapidly progressed to squamous cell carcinoma. In our present study, we found 34 genes with altered DNA methylation in total 124 genes involved in the IL-6 pathway in the UV group. Among these genes with altered methylation, the SOCS1 (suppressor of cytokine signaling 1) gene, which encodes a suppressor in the IL-6-Jak-Stat3 loop, was hypermethylated in the tumor samples.

The top-ranked hypermethylated and hypomethylated genes could enable the discovery of key genes in skin cancer development. For example, RBFOX1 is the top up-regulated gene in terms of methylation status change in UV-irradiated tumors compared with normal epidermis by IPA (Figure 1). RBFOX1 is an RNA-binding protein that is highly expressed in the cytoplasm. RBFOX1 mutations were identified in colorectal cancer cell lines (CRC), and RBFOX1 deletion was observed in a significant proportion of CRC cases (106/419) [31, 32]. However, the role of RBFOX1 in skin cancer development is unclear. Surprisingly, tumor tissues from the DMBA/TPA group exhibited a unique profile in terms of the top 50 genes with up- or down-regulated methylation compared with the profile in the UVB group. Cell adhesion molecule 2 (CADM2) was one of the top methylated genes in DMBA/TPA tumors. CADM2 belongs to a protein family that participates in maintaining cell polarity and that has been considered to be a novel category of tumor suppressors [33]. Clinically, low CADM2 expression predicts a high risk of recurrence in patients with hepatocellular carcinoma after hepatectomy [34]. It has also been reported that aberrant promoter hypermethylation and loss of CADM2 expression are associated with human renal cell carcinoma tumor progression [35]. Our study is the first report to suggest that CADM2 methylation is involved in skin carcinogenesis. Moreover, we identified changes in the methylation patterns of several genes encoding microRNAs, which are also involved in epigenetic regulation. This observation indicates that epigenetic changes may occur at multiple levels with complex crosstalk in skin cancer development and progression.

The genes identified in this study demonstrated significant alterations in response to UV irradiation-induced inflammation and skin cancer development. Although IPA revealed some overlapping signaling changes in response to both UV irradiation and DMBA-TPA treatment and certain highly affected targets were common (including, GRIA1 and TNS1), the top-ranked genes based on fold change differed markedly between the two treatments, indicating that distinct epigenetic mechanisms trigger cancer after exposure to UV or the DMBA carcinogen.

Conclusions

In this study, a comprehensive analysis of the DNA methylation patterns in the UVB or DMBA/TPA induced tumors compared with age-matched normal skin was completed. Genes coding for inflammatory cytokines were identified by IPA to exhibit altered methylation profiles and may be associated with increased susceptibility to tumor development. Specifically, based on changes in methylation, molecular networks were identified that included genes encoding inflammatory cytokines. Additional studies with a particular emphasis on epigenetic alterations, such as DNA methylation, may lead to the development of new strategies for the prevention of skin cancer and inflammation-related skin disease.

Acknowledgments

The authors would like to thank the members of Tony Kong’s laboratory for their helpful discussions. This work was supported in part by institutional funds.

Abbreviations

MeDIP

Methylated DNA immunoprecipitation

UVB

Ultraviolet B

DMBA

7,12-dimethylbenz(a)anthracene

TPA

12-O-tetradecanoylphorbol-1,3-acetate

IPA

Ingenuity® pathway analysis

IL

Interleukin

Footnotes

Conflict of interest statement

The authors declare that there are no conflicts of interest.

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